RNA-Seq Based De Novo Transcriptome Assembly and Gene Discovery of Cistanche deserticola Fleshy Stem

Abstract

Backgrounds Cistanche deserticola is a completely non-photosynthetic parasitic plant with great medicinal value and mainly distributed in desert of Northwest China. Its dried fleshy stem is a crucial tonic in traditional Chinese medicine with roles of mainly improving male sexual function and strengthening immunity, but few mechanistic studies have been conducted partly due to the lack of genomic and transcriptomic resources.ResultsIn this study, we performed deep transcriptome sequencing in fleshy stem of C. deserticola, and about 80 million reads were generated using Illumina pair-end sequencing on HiSeq2000 platform. Using trinity assembler, we obtained 95,787 transcript sequences with transcript lengths ranging from 200bp to 15,698bp, having an average length of 950 bases and the N50 length of 1,519 bases. 63,957 transcripts were identified actively expressed with FPKM ≥ 0.5, in which 30,098 transcripts were annotated with gene descriptions or gene ontology terms by sequence similarity analyses against several public databases (Uniprot, NR and Nt at NCBI, and KEGG). Furthermore, we identified key enzyme genes involved in biosynthesis of lignin and phenylethanoid glycosides (PhGs) which are known to be the primary active ingredients. Four phenylalanine ammonia-lyase (PAL) genes, the first key enzyme in lignin and PhG biosynthesis, were identified based on sequences comparison and phylogenetic analysis. Two biosynthesis pathways of PhGs were also proposed for the first time.ConclusionsIn all, we completed a global analysis of the C. deserticola fleshy stem transcriptome using RNA-seq technology. A collection of enzyme genes related to biosynthesis of lignin and phenylethanoid glysides were identified from the assembled and annotated transcripts, and the gene family of PAL was also predicted. The sequence data from this study will provide a valuable resource for conducting future phenylethanoid glysides biosynthesis researches and functional genomic studies in this important medicinal plant.

The De Novo Transcriptome and Its Functional Annotation in the Seed Beetle Callosobruchus maculatus

“…In our case, single-end reads were few and we therefore only used paired-end reads for the transcriptome assembly. In the end, a total of 474,9 million pairs of reads (96.6%) where retained and used for the assembly [ 35 ].…”

Herba Cistanche (Rou Cong-Rong): One of the Best Pharmaceutical Gifts of Traditional Chinese Medicine

“…(2) For trisaccharide glycosides, there is another glucose or rhamnose at the C6 position of the inside glucose. Recently, Li et al (2015) completed the first deep transcriptome sequencing of the fleshy stem of C . deserticola by RNA-seq and identified some key enzyme genes and pathways that are involved in the biosynthesis of lignin and PhGs, which provides valuable information for this medical plant.…”

Wound Stress, an Unheeded Factor for Echinacoside Accumulation in Cistanche deserticola Y. C. Ma

“…As we know, PAL belongs to a gene family, and there are usually several PAL genes in plant species. This has also been proved in RNA-seq analysis of C. deserticola [ 16 ]. Thus, in this study, the total PAL activity was assayed in nine parts from three plants mentioned in section 3.2.…”

“…The former is derived from l -phenylalanine [ 15 ] and the latter is reported to be derived from tyrosine through l -Dopa or dopamine, which has been proven using an isotope labeled experiment in Olea europaea cell suspension culture [ 15 ]. Besides, it was hypothesized that the phenylethanol group can also be derived from phenylalanine through phenylpyruvate or phenylethylamine, which is concluded from transcriptome analysis of C. deserticola [ 16 ]. In this study, PAL activity was assayed in different tissues from different plant parts.…”

“…The final mixture was mixed vigorously and incubated at RT for 30 min. Absorption at 750 nm (A 750 ) was measured in a microplate reader and recorded for calculation of total phenolic content [ 16 ]. The caffeic acid (Sigma-Aldrich, Shanghai, China) serial solution was used as standard compound and a standard curve between the amount of caffeic acid and A 750 was prepared (Y = 0.8999X − 0.0078, R 2 = 0.9973, linear range: 0.1 to 1.0 mg·mL − 1 ) to calculate the equivalent total phenolic contents in different tissues from different parts.…”